Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes. Also, we have identified the location of the basal promoter of the human NKG2A gene and have shown that it contains a GATA binding site important for its promoter activity. We have shown that GATA-3 is capable of transactivating NKG2A promoter activity and that it is involved in regulating the expression of endogenous NKG2A. Taken together, our data indicate that GATA-3 is an important transcription factor for regulating NKG2A gene expression in NK cells.